Lymphocytes release granules containing serine proteases and perforin membrane pore-forming proteins when they kill virally infected and tumor cells. Isolated perforin-containing granules are cytolytic but require active proteases and calcium to mediate lysis. There are many proteases in the granules, including chymases, tryptases and "Met-ases" which cleave at methionine residues, all implicated in lytic function but all with unknown natural substrates only in the hypothesis is that the proteases cleave their natural substrates only in the presence of calcium and that the natural substrates may include pro-perforins, perforin-promoting proteins, and/or perforin inhibitors. New, specific substrates and inhibitors are needed to address the functions of these newly discovered lymphocyte proteases. In this grant, collaboration between the laboratories of the bioorganic chemist J.C. Powers and the immunologist D. Hudig encompasses four specific aims: (1) to synthesize specific peptide substrates for rat, mouse, and human lymphocyte proteases using computer modeling of cDNA- determined protein sequences to predict appropriate substrates and use the substrates to measure individual proteases released during killing; (2) to design and synthesize more reactive and specific peptide chloromethylketone, peptide phosphonate, and mechanism-based isocoumarin inhibitors for the lymphocyte proteases and to use the inhibitors to probe the mechanism of cell-mediated killing; (3) to synthesize biotinylated and radioactive mechanism-based serine protease inhibitors that block rat RNK- 16 granule-mediated lysis and use them to detect proteases released during killing; and (4) to determine which proteases are required for RNK-16 granule-mediated lysis by using proteases recovered from biotinylated, acylating but not alkylating inhibitors to reconstitute lytic function to inhibited granules. Serine protease control of lymphocyte granule lysis is a new area of protease biology that has implications for specifically targeting and activating perforin proteins as biotherapies for tumors. Inactivation of lymphocyte-mediated lysis also has practical value to block graft rejection and to limit autoimmune diseases, especially after the patient has generated fully differentiated cytotoxic lymphocytes.